Further understanding of the regulation of gene expression depends in part on the ability to accurately measure levels of specific nucleic acid species (DNA and RNA) in defined cell populations. Recent advances in fluorescent cell sorting as well as cell culture technology provide access to homogeneous cell samples with well characterized maturational and/or developmental features.
However conventional methods of analysis of DNA and RNA, such as, for example, by Southern blot hybridization, Northern and "dot blot" hybridization, and even nuclease protection mapping, are not sensitive enough to detect levels of specific DNA or RNA species in many samples when such sample is limited by either low cell number or low copy number per cell. This problem is especially acute in samples of fluorescent cell-sorted populations and in samples of cultured cells. Moreover these methods permit only crude quantitation of the nucleic acid which is present.
In situ hybridization techniques do not solve this problem. In situ hybridization does allow detection of a specific DNA or RNA in single cells but is insensitive and non-quantitative.
Because of its extraordinary high sensitivity, the polymerase chain reaction (PCR) has proven useful in amplifying specific DNAs, including cDNAs, especially those present in low copy number or low abundance (Cohen, S. N., U.S. Pat. No. 4,293,652; Erlich, H. A. et al., EP 258,017; Mullis, K. B., EP 201,184; Mullis et al., EP 200,362; Saiki, R. K., et al., Science 239:487-491 (1988); Mullis, K. B. et al., Meth. Enzymol. 155:335-350 (1987); Scharf, R. K., et al., Science 233:1076-1079 (1986) and Saiki, R. K., et al., Science 230:1350-1354 (1985)).
The polymerase chain reaction amplifies a DNA sequence several orders of magnitude in a few hours. For example, it has been possible to amplify, subclone and characterize low abundance mRNA (Frohman, M. A., et al., Proc. Natl. Acad. Sci. 85:8998-9002 (1988)), and to detect unique mRNA transcripts from abnormal cells in a background of normal cells (Kawasaki, E. S., et al., Proc. Natl. Acad. Sci. 85:5698-5702 (1988), Lee, M-S., et al., Science 237:175-178 (1987)). The use of the polymerase chain reaction, as a DNA diagnostic technique has been recently reviewed (Ladegren, U., et al., Science 242:229-237 (1988)).
The polymerase chain reaction requires the use of oligonucleotide primers complementary to sequences flanking a particular region of interest for primer-directed DNA synthesis in opposite and overlapping directions. With repeated cycles of high-temperature template denaturation, oligonucleotide primer reannealing, and polymerasemediated extension, DNA sequences can be faithfully amplified several hundred-thousand fold.
Generally PCR requires knowledge of the sequence of both the 5' and the 3' end of the template being amplified so that two different primers for each template may be designed, one primer for the sense strand and one primer for the antisense strand. However, it is known in the art to amplify nucleic acid targets with only one primer, using "anchored PCR," wherein it is necessary only to know the sequence of the 3' end of the target. Loh, E. Y. et al., Science 243: 217 (1989).
In theory, only one copy of the target gene need be present in a sample for the polymerase chain reaction to adequately target and amplify it. For example, the polymerase chain reaction amplification technique has been used to analyze the DNA in an individual diploid cell and a single sperm. Li, H. et al., Nature 335: 414-417 (1988).
Although it has been possible to detect and amplify large amounts of rare DNA or mRNA transcripts, it has been more difficult to quantitate the amount of the nucleic acid species in the starting material for, although PCR can detect the presence of a targeted nucleic acid species in the starting material, the results of conventional PCR cannot be used to calculate the pre-amplification levels of that targeted nucleic acid. This has precluded the use of PCR in many situations, for example, in an analysis of the fold induction of a specific mRNA in response to exogenous stimuli.
The main constraint in obtaining quantitative data from conventional PCR is inherent in the amplification process. Because amplification is (at least initially) an exponential process, small differences in any of the variables which control reaction rate will dramatically affect the yield of PCR product. Variables which influence the rate of the PCR reaction include the concentrations of polymerase, deoxynucleoside triphosphate substrates (dNTP's), Mg.sup.++, target DNA and primers; annealing, extension and denaturing temperatures; cycle length and cycle number; the rate at which the temperature is changed from one step to another within each amplification cycle; rate of "primer-dimer" formation; and presence of contaminating DNA.
Further, even when these parameters are controlled precisely, there is tube-to-tube variation which precludes accurate quantitation. For example, significant differences in yield occur in PCR samples which are prepared as a pool and then aliquoted into separate tubes and amplified in the same run. The basis for this variation is not certain--it may be related to events which occur during the first few cycles, or small temperature variances across the thermal cycler block.
Methods have been described for quantitating cDNA species by PCR, usually by co-amplifying a second, unrelated template (Rappolee, D. A., et al., Science 241:708-712 (1988)). These methods are critically dependent on several variables, including cycle number and amount of starting mRNA of each species. Even when these variables are adequately controlled, it is unlikely that the unrelated control template will be amplified at precisely the same rate as the unknown template. Small differences in the rate of amplification of the two templates are magnified during PCR and may grossly over- or underestimate the amount of the unknown template present.
For example, Chelly et al., Nature 333:858-860 (1988), attempt to overcome the above limitations of PCR in calculating pre-amplification levels of dystrophin mRNA by co-amplifying the target's mRNA with that of a reporter mRNA, aldolase A, and relating the fold amplification of the reporter molecule to that of the target. This method does give an approximation of the amount of a starting mRNA in the sample. However, Chelly et al.'s, method requires knowing primer sequences for two different targets and does not overcome differences in the rates of primer-dimer formation between the two sets of primers.
Although this approach has been successfully applied in several systems, data must be obtained at early cycle number (e.g.&lt;20 cycles) when efficiency of amplification is a "constant". This often necessitates blotting of PCR products with labeled probe, with difficulties in quantitation inherent in blotting techniques, and is difficult to apply to low abundance mRNA from small numbers of cells.
Thus a need still exists for a method which adapts the sensitivity of PCR technology to quantitative analysis of the nucleic acid species being amplified.